The present invention concerns the human hepatitis B virus genome, isolated from hepatocellular carcinoma (HCC), and with a mutation at amino acid residue 133 (Methionine to Threonine) within the major surface antigen, its nucleotide sequence, the deduced amino acid sequence of the four major proteins, antigen, antibody, detection systems, development of effective vaccines, and antiviral agents.
HCC is one of the most common human liver cancers, particularly in Asia where 70% of the worldwide new cases are found every year. It usually arises in cirrhotic livers as a complication of chronic liver disease. The clinical manifestations of HCC patients are unspecific with signs and symptoms only appearing in the later stages of the cancer.
One of the major causes of chronic liver diseases is hepatitis B viral infection. First discovered in 1963 as a human virus that is transmitted parenterally, chronic hepatitis B viral infection has been most commonly implicated in serological undefined pathogenesis of HCC. Despite the fact that hepatitis B virus does not display features of a complete viral oncogene, its involvement in the development of HCC can be attributed to various aspects of its interaction with host hepatocyte cells. These include the promiscuous transcriptional activity of the smallest viral protein, X, which enhances the expression level of many cellular target genes including proto-oncogenes. On the other hand, integrated viral DNA in the host chromosomes is regularly found in HCC patients. There is also evidence for an active role in the development of HCC by the major surface antigen. This protein has served as the main detection marker for carriers of hepatitis B virus. The most antigenic epitope is a highly conserved region spanning 23 amino acid residues and located from amino acid position 124 to 147 of the major surface antigen. This small region designated as the group specific determinant “a” is found in all subtypes and isolates of hepatitis B viral genomes. Its antigenic properties seem due to its proposed double loop structure, to which the vaccine-induced neutralizing antibody binds.
Our epidemiological data indicate that the wild type major surface antigen has been found in most HCC patients. Furthermore, observation indicates that several variants of the major surface antigen from HCC patients may be involved in the pathogenesis of HCC. Direct sequencing analysis indicated that 24 out of 63 HCC patients (around 38%) carry various mutations in the “a” epitope of the major surface antigen. When both the wild type and variant cases are combined, the proportion of the variant virus carrying a mutation at amino acid residue 133, located in the first loop of the “a” epitope of the major surface antigen (Methionine to Threonine), is as high as 12.7% in 63 HCC patients from the Southeast Asia region, and present in 5 local HCC cases. However, the same mutation is found in only 2% of hepatitis B virus carriers in a random population (more than 100 cases). The significance of this variant at amino acid residue 133 is further strengthened by the fact that the proportion of variant virus at amino acid residue 145 (Glycine to Arginine), better known as a vaccine-induced mutant and located in the second loop of the “a” epitope, remains constant at 8% in hepatitis B virus carriers in the random population sample.
Although this variant hepatitis B viral strain, carrying a mutation at amino acid residue 133 (Methionine to Threonine) of the major surface antigen in HCC patients, may arise differently from those induced following vaccination (i.e. with a mutation at amino acid residue 145 of the major surface antigen), this strain shares similar characteristics in that both are stable and cases of vertical transmission of these strains have been reported, despite effective hepatitis B virus prophylaxis and hepatitis B immunoglobulin (HBIG) being used.
The emergence of the variant human hepatitis B virus, carrying mutations in the “a” epitope of the major surface antigen, in HCC is of concern. The high proportion of the mutant virus with a substitution at amino acid residue 133 of the major surface antigen is of particular interest as it may point to a close association with the pathogenesis of HCC. This correlation would therefore require the urgent development of specific detection systems as well as effective prophylactic and therapeutic vaccines and antiviral agents. Determination of the nucleotide sequence of this mutant virus constitutes the first step towards these aims and will certainly be helpful for developing the above-mentioned diagnostic and treatment schemes.